CN106868004B - Site pair and primer pair capable of being identified and acted by LR enzyme and plasmid construction method - Google Patents

Abstract

The invention relates to a site pair capable of being identified and acted by LR enzyme, the sequence of which is composed of oligonucleotide 1 shown in SEQ ID NO. 1 and oligonucleotide 2 shown in SEQ ID NO. 2. Also relates to a primer pair, which is characterized by consisting of a primer 1 and a primer 2, wherein the primer 1 comprises an oligonucleotide 1 and a characteristic primer at the 5 ' end of a gene to be amplified, the primer 2 comprises an oligonucleotide 2 and a characteristic primer at the 3 ' end of the gene, and the oligonucleotide 1 and the oligonucleotide 2 are positioned at the 5 ' end of the corresponding primers. Also relates to a plasmid construction method using the primer pair. The invention shortens the recognition site of LR enzyme in LR reaction to about 36BP, greatly improves the original Gateway cloning, can bypass BP reaction in the construction process of expression plasmid, directly carries out LR reaction, simplifies operation steps and saves labor cost and reagent cost.

Description

Site pair, primer pair and plasmid construction method that can be recognized and acted by LR enzyme

technical field

The present invention relates to the field of molecular biology, more particularly, to a pair of loci that can be recognized and acted on by LR enzymes, a pair of primers comprising the pair of loci, and a plasmid construction method using the pair of primers.

Background technique

Gateway is the name of a vector construction method that requires two reactions through BP and LR. BP reaction requires BP enzyme, LR reaction requires LR enzyme, BP reaction is to connect the target DNA fragment to the intermediate vector, and LR reaction is to transfer the target fragment connected to the intermediate vector to the final vector. The BP reaction is the recognition exchange of attB and attP specific sites to generate attL (the carrier is called entry vector) and attR specific sites, and the LR reaction is the recognition exchange of attL and attR specific sites to generate attB and attP specific recognition sites. attL is generally 125bp. We need to add 25bp (bp is the number of bases) attB site to the 5' ends of the left and right primers of the target DNA fragment. There is an attP (the vector is called pDONR vector) recognition site on the intermediate vector. There is an attR recognition site.

At present, most of the laboratories that use Gateway technology to construct vectors are mainly constructed in the following two ways:

First, the DNA target fragment is ligated to the pDONR vector through BP reaction to obtain the entry vector, and then the entry vector reacts with the final vector through LR to ligate the target fragment to the final vector. However, the BP enzyme is expensive, and in practical applications, due to the low quality of the recovered PCR products, the concentration requirements cannot meet the requirements of the BP reaction, so this step is a bit difficult.

Second, the DNA fragment is ligated to the entry vector by enzyme cleavage, and then the entry vector reacts with the final vector through LR to ligate the target fragment to the final vector. This method connects the vector by enzyme cleavage, which abandons the advantages of fast and high efficiency of recombination site connection, and the connection direction of the single-enzyme-cleaved DNA fragment in the enzyme-cleavage ligation method cannot be determined. Enzymes are highly efficient.

In 2008, the Monsanto Company in the United States published an article that attL sites were directly synthesized at the 5' ends of the left and right primers of the DNA fragments, and then the PCR product was subjected to an LR reaction with the final vector to connect the target fragment to the final vector. The original length of attL of 125bp was shortened to 48bp, and this method has not yet been applied in the laboratory. This method still has defects. The 48 bp added to the 5' ends of the left and right primers of the DNA is too long, and the cost of synthesizing the primers increases. In addition, during PCR amplification, because the primers are too long, the DNA fragments must be amplified by a two-step method, which is troublesome and also Increased the cost of synthesis of repetitive primers.

In 2013, life company in the United States also published an article that carried out BP and LR reactions together, and this method is rarely used in laboratories. Although this method reduces the experimental steps, the BP reaction and the LR reaction still need to be carried out, and the cost of the reagents for the BP reaction is relatively expensive.

SUMMARY OF THE INVENTION

In order to solve the above problems, the present invention provides a pair of sites that can be recognized and acted on by LR enzymes, which consists of oligonucleotide 1 whose sequence is shown in SEQ ID NO: 1 and an oligonucleotide whose sequence is shown in SEQ ID NO: 2. Consists of 2 nucleotides.

The present invention also provides a primer pair, which consists of primer 1 and primer 2. Primer 1 includes oligonucleotide 1 whose sequence is shown in SEQ ID NO: 1 and a characteristic primer at the 5' end of the gene to be amplified. Primer 2 The oligonucleotide 2 with the sequence shown in SEQ ID NO: 2 and the 3' end characteristic primer of the gene are included, and both oligonucleotide 1 and oligonucleotide 2 are at the 5' end of the corresponding primer.

The present invention also provides a kind of plasmid construction method, it comprises the following steps:

S1: use the primer pair described in claim 2 or 3 to amplify the gene to be amplified to obtain a PCR product;

S2: The PCR product is subjected to LR reaction with the final plasmid with attR site to obtain the final plasmid with the gene.

Preferably, the final plasmid described in S2 is an expression plasmid with a selectable marker, and S2 specifically includes:

S21: mixing the PCR product, the final plasmid and the LR reaction reagent, and performing an LR reaction to obtain a reaction product;

S22: transform competent Escherichia coli cells with the reaction product;

S23: use the selectable marker to screen the E. coli cells transformed with the expression plasmid carrying the gene;

S24: culturing the E. coli cells screened in S23, and extracting the plasmid to obtain a purified expression plasmid with the gene.

Preferably, the E. coli cells are DH5α.

Preferably, the selectable marker is antibiotic resistance.

The invention shortens the recognition site of the LR enzyme in the LR reaction to about 36bp, greatly improves the original Gateway clone, so that in the process of constructing the expression plasmid, the BP reaction can be bypassed, and the PCR product and the final plasmid can be directly used for the LR reaction , simplifies the operation steps and saves labor costs and reagent costs.

Description of drawings

Fig. 1 is the principle schematic diagram of direct LR reaction;

Fig. 2 is the plasmid map of R4L1pDEST_HISi;

Fig. 3 is the electrophoresis photograph of colony PCR of attL"-400+R4L1pDEST_HISi;

Figure 4 is an electrophoresis photograph of colony PCR of attL"-800+R4L1pDEST_HISi.

Detailed ways

The principles and features of the present invention will be described below with reference to examples. The examples are only used to explain the present invention, but not to limit the scope of the present invention.

In the present invention, the traditional attL-specific site pairs of about 125bp are shortened to about 36bp each, and the site pairs are named as attL" (wherein, the attL"1 sequence is shown in SEQ ID NO: 1, and the attL"2 sequence is shown in SEQ ID NO: 1. As shown in SEQ ID NO: 2). The PCR product was then inserted directly into the final vector by LR reaction.

In this experiment, DNA fragments of 400bp and 800bp were used to connect with the R4L1pDEST_HISi vector to verify the attL of about 36bp. Among them, the plasmid map of R4L1pDEST_HISi is shown in Figure 2. The steps are as follows:

1. PCR amplification of the target fragment

Use primers 1 (SEQ ID NO: 3) and 2 (SEQ ID NO: 4) to amplify a 400 bp long target fragment and primers 3 (SEQ ID NO: 5) and 4 (SEQ ID NO: 6) to amplify an 800 bp long target Fragment, 15μL amplification system consists of: template 0.5μL, 10×Taq buffer 1.5μL, 2.5mM dNTP 0.5μL, primer 1/20.5μL/0.5μL, Taq DNA polymerase 0.4μL, and the rest is ddH ₂ O.

The reaction program was: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, 72°C at 1000 bp/min, 31 cycles; extension at 72°C for 5 min; 25°C for 10 min.

After PCR, run electrophoresis with 1% agarose, and recover the target PCR product according to the gel recovery kit (Sangon Bioengineering Co., Ltd., Shanghai, China).

3. LR response

The recovered PCR product was directly subjected to LR reaction with R4L1pDEST_HISi vector. The system and conditions of the LR reaction are shown in Table 1.

Table 1 LR reaction system

4. Transformation of DH5α and Antibiotic Screening

After the LR reaction, the reaction solution was transferred into DH5α competent cells (Quanjin Biotechnology, Beijing, China). (1) Take out the competent cells from the refrigerator at -80°C and put them on ice for 5 minutes, after the competent cells are melted; (2) All the reaction solution is added to the competent cells, and ice-bathed for 30 minutes; (3) Quickly remove the competent cells The mixture was water bathed at 42°C for 90s; (4) The mixture was taken out and ice bathed for 3 minutes; (5) 800 μL of LB liquid medium was added, and cultured at 37°C at 180 r/min for 45 minutes; (6) The culture was incubated at 7000 r/min for 1 minute Centrifuge, remove 700 μL of supernatant, use the remaining 100 μL of LB medium to mix the precipitate with a gun suction, spread 100 μL of bacterial liquid evenly on the petri dish containing the corresponding antibiotics with a coating stick, and cultivate in a 37 °C incubator for 12 h .

5. Colony PCR Validation

After 12 hours, the grown colonies were selected as templates for colony PCR. The system was the same as before, and the primers used and the extension time corresponded to the PCR conditions for amplifying DNA fragments. The results are shown in Figures 3 and 4: Figure 3 is the colony PCR of attL"-400+R4L1pDEST_HISi, in which the target band appeared in lanes 6, 10 and 12, and 47880 is the positive control; Figure 4 is the attL"-800+R4L1 pDEST_HISi Colony PCR, in which the target band appeared in lanes 1, 2, 3, 4, 5, 6 and 8, T-800 was the positive control, and ddH ₂ 0 was the negative control.

It can be seen that this method can directly perform the LR reaction on the PCR product, and obtain the desired clone.

The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

sequence listing

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Claims (6)

1. The application of the DNA fragment as the site pair capable of being identified and acted by the LR enzyme is characterized in that the DNA fragment consists of oligonucleotide 1 with a sequence shown as SEQ ID NO. 1 and oligonucleotide 2 with a sequence shown as SEQ ID NO. 2.

2. A primer pair is characterized by consisting of a primer 1 and a primer 2, wherein the primer 1 consists of an oligonucleotide 1 with a sequence shown as SEQ ID NO. 1 and a characteristic primer at the 5 ' end of a gene to be amplified, the primer 2 consists of an oligonucleotide 2 with a sequence shown as SEQ ID NO. 2 and a characteristic primer at the 3 ' end of the gene, and the oligonucleotide 1 and the oligonucleotide 2 are positioned at the 5 ' end of the corresponding primers.

3. A plasmid construction method is characterized by comprising the following steps:

s1: amplifying the gene to be amplified by using the primer pair of claim 2 to obtain a PCR product, wherein the length of the gene to be amplified is 400-800 bp;

s2: and carrying out LR reaction on the PCR product and a final plasmid R4L1pDEST _ HISi, and screening to obtain a final plasmid with the gene, wherein the final plasmid is provided with an attR site pair, and ccdB genes are arranged between the attR site pair.

4. The method according to claim 3, wherein the final plasmid in S2 is an expression plasmid with a selection marker, and S2 specifically comprises:

s21: mixing the PCR product, the final plasmid and an LR reaction reagent, and carrying out LR reaction to obtain a reaction product;

s22: transforming competent E.coli cells with said reaction product;

s23: screening Escherichia coli cells transformed with an expression plasmid carrying the gene using the screening marker;

s24: and (4) culturing the Escherichia coli cells obtained by S23 screening, and extracting plasmids to obtain purified expression plasmids with the genes.

5. The method of claim 4, wherein the E.coli cell is DH5 α.

6. The method of claim 4, wherein the screening marker is resistant to antibiotics.

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